Download ECCell_D6_1 Demonstration of sequence

Project no. 222422
Project acronym: ECCell
Project title: Electronic Chemical Cell
Instrument:
STREP/FET OPEN
Thematic Priority: Theme 3 Information and Communication Technologies
Deliverable 6.1: report, public (PU)
Demonstration of sequence-specific DNA processing with programmable ECCell gels.
Due date of deliverable: 1. 11. 2010
Actual submission date: 03. 11. 2010
Start date of project: 1.09.2008
Organisation name of lead contractor for this deliverable:
Ruhr-Universität Bochum (RUB-BioMIP), John McCaskill
Duration: 3 years
9. Deliverable n. 6.1: Demonstration of sequence-specific DNA
processing with programmable ECCell gels.
9.1 Introduction
During the course of the ECCell project, the consortium leader RUBa, who is dealing with
microfluidic devices as the electronic scaffold for the electrochemical cell, made some important
findings. The triblock copolymer Pluronics, which has the molecular structure poly(ethylene
oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-b-PPO-b-PEO), was employed as
a gel matrix within microfluidic channels to separate ODNs of various lengths. The striking
feature of this material to other gel matrices is that it can be reversibly dissolved within the
microfluidic device by electronically induced heating to a sol state. In this way, ODN separation
can be carried out in the gel state and subsequently different ODNs can be directed to different
positions within the microfluidic channels after dissolution of the gel by travelling wave
electrophoresis. This is a significant step forward to realize an electronically controlled cell
because it allows performing anabolic reactions and separation of the resulting products.
However, since the genetic material of the cell is oligo- and polynucleotides one obstacle is
remaining, i.e., the separation of equally long, single stranded (ss) DNA sequences that usually
exhibit the same electrophoretic mobilities.
In the following, three different materials are described, with which this goal was successfully
realized. It is well known that ssDNA sequences can be specifically retained when the
complement is covalently connected to the gel matrix. However, the resulting gels (mostly
polyacrylamide) are not reversible. We decided to generate soft matter DNA nanoparticle
systems that due to their size and molecular interactions are retained in the reversible Pluronics
matrix.
This allows the development of sequence-specific programmable gel processing as an
application of the technology being developed in ECCell. We commence this report on the
deliverable with section son the synthesis and characterization of the new materials and then
with microchannel experiments (via CGE and chemical microprocessors) on their use to program
sequence-specific separations of DNA.
9.2 DNA block copolymer micelles stabilized by formation of a semiinterpenetrating network in the core
DNA-b-PPO micelles were combined with Pluronics to form blended micelles. In this case a
strong molecular interaction between the DNA particles and the Pluronics gel matrix should be
achieved. For stabilization of the particles a polymernetwork was generated in the core of the
micelles. The stabilization reaction was carried out by internalization of pentaerythrytol
tetraacrylate (PETA) in the micelles and UV induced cross-linking. Figure 32 gives a schematic
representation of the architecture of the blended DNA block copolymer aggregates.
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Figure 32: Schematic of the mixed micelle architecture and chemical structures of the polymeric components. (A) PEO block of
Pluronic. (B) DNA block of DBC. (C) PPO blocks of Pluronic or DBC. (D) and (E) Probes at 5’- and 3’-ends of the
complementary DNA, respectively. (F) Hydrophobic compound loaded into the hydrophobic core. (G) Cross-linked
nanodomains of PETA in the core.
The stabilization of the micelles by forming a semi-interpenetrating network in the core that
contained Pluronics F127 and DNA-b-PPO in a ratio of 5:1 was assessed by incorporation of
pyrene and cooling below the critical micelle temperature (CMT). The fluorescence
spectroscopic analysis revealed that core cross-linked micelles retained the pyrene in the core
while non cross-linked blended micelles released the payload and disassembled into their
individual components. In a next step it needed to be demonstrated that the cross-linked blended
micelles are still able to hybridize with complementary DNA. The ability of the aggregates to
undergo Watson-Crick base pairing was successfully proven with FRET experiments employing
fluorophore labelled Pluronics F127 and labelled ODNs. Additional proof for the ability of the
blended micelles to hybridize was achieved with ODN-labelled gold nanoparticles and
subsequent TEM analysis. Moreover, the particles were analyzed by AFM and dynamic light
scattering (DLS) (see A. Herrmann et al, Chem Commun. 2010, 46, 4935 for further
information).
9.3 DNA block copolymer micelles containing more hydrophobic polymer cores.
Several PIs (Mw = 950, 2810, 4710, and 7630 g/mol) with a terminal hydroxyl group were
prepared by anionic polymerization. Subsequently, the semitelechelic polymers were converted
into the corresponding phosphoramidites (Figure 33A) and coupled to ODNs present on the solid
support to form DNA-b-PI in excellent yields (Figure 33B and 33C).
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Figure 33: A) PI-phosphoramidite, B) Sequence composition of DNA-b-PI and C) Gel-electrophoretic analysis of the crude
product of DNA-b-PI demonstrating the high efficiency of the coupling reaction on the solid support.
After micellisation the corresponding aggregates were analyzed by AFM and DLS revealing
particles with a diameter of 23 + 3 nm. Again hybridization experiments with these selfassembled structures proved to be successful.
9.4 DNA nanoparticles stabilized by hydrophobic chains attached to the
nucleobase.
As a third alternative to produce DNA nanoparticles that can retain DNA in a reversible
Pluronics gel matrix aggregates were produced that are composed of ODNs which are
functionalized with hydrophobic chains at the nucleobases. For that purpose hydrophobicity was
imparted by the introduction of a dodec-1-yne chain at the 5-position of the uracil base, which
allowed precise and simple tuning of the hydrophobic properties through solid-phase DNA
synthesis. Figure 34 gives an overview of the employed materials including the phosphoramidite
building block, the incorporation into ODNs and three different architectures with their
corresponding hybridization products.
The micelles formed from these modified DNA sequences were characterized by atomic force
microscopy, dynamic light scattering, and polyacrylamide gel electrophoresis (see A. Herrmann
et al, Chem. Eur. J. 2010, DOI: 10.1002/chem.201001816.) These experiments revealed the role
of the quantity and location of the hydrophobic units in determining the morphology and stability
of the micelles. The effects of hybridization on the physical characteristics of the DNA micelles
were also studied; these results showed potential for the sequence-specific noncovalent
functionalization of the self-assembled aggregates and their utilization in the microfluidic
devices.
Figure
34: Synthetic scheme and representation of lipid–DNAs. a) The precursor, 5-(dodec-1-ynyl)uracil
deoxyribophosphoramidite (left) was used in conventional solid-phase DNA synthesis (center), and deprotection
yielded the lipid–DNA (right). b) Schematic representation of the ss and ds lipid–DNA amphiphiles (U2M, U2T, and
U4T) investigated.
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9.5 Testing the ability of DNA aggregates to retain DNA in reversible gels (RUG)
After preparation we tested the ability of the DNA aggregates to sequence specifically retain
DNA in conventional gels that can be disaggregated after electrophoresis, i.e. agarose and
Pluronics gels.
Figure 35: Electrophoretic analysis of DNA aggregates vs. non-modified DNA in agarose. The relative mobilities in the table
were calculated in respect to ssU4T and ssDNA as slowest and fastest moving species, respectively.
In Figure 35 the electrophoretic analysis of the mobility of DNA aggregates in an agarose gel are
summarized. These results clearly show that all the DNA polymer and DNA alkyl modified
materials exhibit significant less electrophoretic mobility than non-modified DNA. All
hydridized samples, as expected, migrate less than their corresponding double stranded
counterparts. Finally, the 12mer ODN functionalized with four hydrophobic chains (U4T)
exhibited an extremely low electrophoretic mobility compared to the other aggregates. Very
similar results were obtained in experiments where Pluronics acted as a gel matrix. Figure 27
shows the migration of polymeric DNA aggregates in the Pluronics gel. Pristine DNA-b-PPO
micelles (lane 2) and DNA-b-PI micelles (lane 3) migrate significantly slower than the ds DNA
control. Blended micelles (lane 4) seem to disaggregate upon longer running times.
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Figure 36: Gel electrophoretic analysis of DNA polymer aggregates in a Pluronics matrix.
9.6 Demonstration of programming containment via sequence specific DNA
retention with ECCell gels in CGE (RUBa)
As the separation matrix we used the same hydrogel material (Pluronic® F127) as successfully
tested in the chemical microprocessor chip in the first project year. The advantage in terms of
microfluidic integration is, that these kinds of hydrogel monomers are reversible thermoresponsive gels. At low temperatures, the dissolved materials acts as a free flowing solution
while at elevated temperatures self assembled micelles form a quasi-lattice with a face-centered
cubic structure leading to an increase in viscosity and hence to the formation of a gel.
Furthermore, Pluronic® F127 is known to be a good separation medium for electrophoresis.
Figure 37: (A) Linear electrophoretic DNA-transport of a reference Oligo31 3’-GGAGCGAGACGATTAGGACAAT5‘Alexa488 (22-mer) and Oligo30 Atto488-5'-GAA TCC GCA AAA-3' (cDNA) in a capillary as well as (B) a
hybridised solution of ssU4T + cDNA (dsDNA-b-copolymer). CGE conditions: T = 30°C, pH 7.5, TBE running
buffer, 600 V (56 µA), injection 3 sec., C = 2*10-6 M.
The CGE pre-experiments shown in figure 37 were designed to give us information about (A)
the mobility of the cDNA in comparison with a reference ssDNA (22mer) and (B) the duplex of
scp-DNA and the complementary cDNA in Pluronic F127. The hydridized duplexes migrate less
than their corresponding single stranded counterparts, and the 22mer reference ssDNA, and show
a diffuse peak signal. To find out the best conditions for integration in the microfluidic
environment of the various modified DNA oligomers, especially the one functionalized with four
alkyl groups (ssU4T 10 as synthesized by the group of RUG, see also WP2 & 4) capillary gel
electrophoresis (CGE) analysis was performed for the modified oligonucleotides.
To test the sequence specific retention the cDNA (Oligo30) as well as a non matching sequence
(Oligo 03) was injected in a hydrogel mixture containing Pluronic F127 and the 12mer DNA
functionalized with four hydrophobic chains (ssU4T 10) as shown in figure 38. The result is, that
the non-complementary strand moves as a single strand through the gel whereas the
complementary oligonucleotide forms the duplex with the scp-DNA in combination with a
significant reduced electrophoretic mobility.
A movie regarding sequence specific DNA migration by immobilized DNA-b-copolymers in
CGE-capillaries (CGE_scpDNA_retention.mov) as a part of the Devireable 4.2 is shown in the
download section for reviewers at the ECCell homepage.
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Figure 38: Sequence specific DNA retention of Oligo30 Atto488-5'-GAA TCC GCA AAA-3' (cDNA) in a CGE experiments in
comparison to non-matching DNA. CGE conditions: T = 30°C, pH 7.5, TBE running buffer, 600 V (88 µA), injection
3 sec., C = 2*10-6 M.
9.7 µ-fluidic integration experiments of sequence specific DNA retention in
ultra-thin hydrogel films using the chemical microprocessor
Besides the investigation of the mobility using CGE we also tested the system in ultra-thin
hydrogel films on-chip. The preparation procedure is shown in figure 39. The chip was treated
with tiny droplets of a cold mixture (5°C) of Pluronic F 127 and 12mer scpDNA (ssU4T 10)
(mixing ratio = 100:1) as well as the same mixture containing non-matching DNA (2*10-6 M
Alexa488-5'-TTC GAT GAT GCG C-3'). The samples were heated up to 30°C to trigger gelation
embedded in a pure Pluronic F127 hydrogel matrix. Finally the samples were covered by a thin
glass slide.
Figure 39: Left: Illustrated procedure to produce ultra-thin hydrogel films. Right: Schematic presentation of sequence specific
DNA retention in ultra-thin hydrogel films.
Regarding the results of the CGE, the non-complementary DNA should not interact with the scpDNA and therefore they were concentrated if the electrodes are activated (positive or negative
voltage) and transported using the electric fields inside the hydrogel matrix as shown in figure
40.
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Figure 40: The movie series show that negative charged electrodes repel the non-complementary strands. Positive charged
electrodes lead to a concentration of the non-matching species. Experimental conditions: 30°C, 25% w/v Pluronic F
127. Excitation wavelength: 488 nm.
The control experiment performed in an embedded scpDNA/Pluronic spot without the noncomplementary strands shows no interactions with the microelectrodes except for a low
concentration of non-hybridised scpDNA species outside of the gel-spot, which diffused inside
the pure Pluronic F127 matrix (figure 41).
Figure 41: The movie series show or minor interactions with the microelectrodes respectively. Experimental conditions: 30°C,
25% w/v Pluronic F 127. Excitation wavelength: 488 nm.
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